Vitamin D analogs and methods of preparing these compounds

ABSTRACT

The invention relates to vitamin D analogs of general formula (I) wherein: R 1  is a hydrogen atom or a substituent selected from the group consisting of hydroxy, CH 2 OH, CH 2 CH 2 OH, CH 2 CH 2 CH 2 OH, OCH 3 , OCH 2 OH, OCH 2 CH 2 OH and OCH 2 CH 2 CH 2 OH; R 2  is a hydrogen atom or a substituent selected from the group consisting of OCH 3 , OCH 2 OH, CH m (CH 2 OH) n (CH 2 CH 2 OH) p , (CH 2 ) q OH and O(CH 2 ) r OH; wherein: m is 0 or 1, p, q and n are 0-3, r is 1-3 and m+n+p=3; with the proviso that R 1  and/or R 2  contain at least one OH group; R 3  is a straight or branched, saturated or unsaturated aliphatic hydrocarbon of 6-13 C atoms which may be substituted with one or more substituents from the group hydroxy or fluoro. The invention further relates to methods of preparing these compounds, a pharmaceutical composition containing these compounds and to their use in pharmacotherapy and cosmetics.

This application is a 371 of PCT/EP97/02429 filed May 2, 1997.

The invention relates to new vitamin D analogs, to methods of preparingthese compounds and to their use in pharmacotherapy and cosmetics.

It is generally known, that vitamin-D compounds or vitamin-D relatedcompounds (“vitamin-D analogs”) have a strong biological activity andmay be used in all those cases in which problems with the calcium andbone metabolism play a part. A few years ago it was found that variousactive vitamin-D compounds and analogs also have otherpharmacotherapeutic activities and may be used successfully, forexample, for the treatment of certain skin and bone diseases, forcosmetic applications and for treating diseases which are related tocell differentiation, cell proliferation or imbalance in the immunesystem, including diabetes mellitus, hypertension and inflammatorydiseases such as rheumatoid arthritis and asthma. In addition, thesecompounds may be used in various veterinary applications, and fordiagnostic purposes.

Further it is known from J. Med. Chem. 38 (1995), 4529-4537 that 1 α,25-dihydroxy vitamin D₃ analogs of the formula

wherein S₁ is OH, HOCH₂— or HOCH₂CH₂ have very low affinities, i.e. nomore than 10⁻³ the affinity of calcitriol, for the calf thymus vitamin Dreceptor, and considerable antiproliferative activities in murinekeratinocytes.

Surprisingly it has now been found that a group of new vitamin D analogshas an activity of up to 6 times that of calcitriol, i.e. is much moreactive than the above mentioned known analogs, in the same test(affinity for the calf thymus vitamin D receptor).

The present invention relates to compounds of the general formula

wherein

R₁ is a hydrogen atom or a substituent selected from the groupconsisting of CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH, OCH₃, OCH₂OH, OCH₂CH₂OH andOCH₂CH₂CH₂OH;

R₂ is a hydrogen atom or a substituent selected from the groupconsisting of OCH₃, OCH₂OH, CH_(m)(CH₂OH)_(n)(CH₂CH₂OH)_(p), (CH₂)_(q)OHand O(CH₂)_(r)OH;

wherein:

m is 0 or 1, p, q and n are 0-3, r is 1-3 and m+n+p=3;

with the proviso that R₁ and/or R₂ contain at least one OH group;

R₃ is a straight or branched, saturated or unsaturated aliphatichydrocarbon of 6-13 C atoms which may be substituted with one or moresubstituents from the group hydroxy or fluoro.

The above new vitamin D analogs of the invention, presented by thegeneral formula I, are valuable substances. The biological results, asillustrated in the Examples, indicate that these compounds are promisingas biologically active substances and may be used in all above-mentionedpharmacotherapeutic indications, more in particular for the treatment ofosteoporosis, renal osteodystrophy, osteomalacia, skin disorders such aspsoriasis (and other hyperproliferative skin diseases), eczema anddermatitis, myopathy, leukaemia, breast and colon cancer, osteosarcomas,squamous cell carcinomas, melanoma, certain immunological disorders, andtransplant rejections.

Furthermore, the new vitamin D analogs of the invention may be used forwound healing and may be incorporated in cosmetic compositions, such ascreams, lotions, ointments and the like, in order to preserve, conditionand/or protect the skin and to improve various skin conditions, such aswrinkles, dry skin, skin slackness and insufficient sebum secretion. Thenew vitamin D analogs may also be used for diagnostic purposes.

Preferred compounds of the general formula (I) are the compounds whereinR₃ is the group —(CH₂)₃—C(CH₃)₂OH.

Especially preferred are the compounds of formula (I) wherein R₃ is thegroup —(CH₂)₃—C(CH₃)₂OH and wherein:

a) R₁ is a hydrogen atom and R₂ represents CH₂CH₂OH, CH₂CH₂CH₂OH,CH(CH₂CH₂OH)₂, C(CH₂CH₂OH)₃ or C(CH₂CH₂OH)₂CH₂OH; or

b) R₁ is OH, CH₂OH or (CH₂)₂OH and R₂ represents hydrogen, OH, OCH₃, orO(CH₂)_(r)OH, wherein r has the value 1 to 3.

Most particularly preferred are the compounds of formula (I) wherein R₃is the group —(CH₂)₃—C(CH₃)₂OH and wherein:

R₁ is hydroxy, CH₂OH and CH₂CH₂OH and R₂ represents hydrogen, CH₂OH orCH₂CH₂OH.

It is a special merit of the present invention that the above newvitamin D analogs can easily be prepared from readily available startingmaterials.

Consequently, the invention also relates to a method of preparing avitamin D analog of the general formula I, as defined above, whichmethod is characterized in that a compound of the general formula

wherein:

R₃′ is a straight or branched, saturated or unsaturated aliphatichydrocarbon of 6-13 C-atoms which may be substituted with one or moresubstituents from the group protected hydroxy or fluoro, is reacted withan organometallic compound of the general formula

R₁′ and R₂′ are the hydroxy protected analogs of R₁ and R₂ definedabove, X is Li,MgCl, MgBr or MgI, to yield a compound of the generalformula

followed by dehydration and deprotection of the hydroxy group(s).

The compounds having formula I can also be obtained by reacting acompound of the general formula

wherein R₃′ has the meaning given above, and R₄ is a protected hydroxygroup, with a compound of the general formula

wherein X′ is halogen, preferably Br, and R₁′ and R₂′ have the abovegiven meanings, under the influence of an alkyl lithium compound, a zinchalogenide, preferably ZnCl₂, and a transmetallation catalyst, followedby deprotection of the hydroxy group(s). An example of atransmetallation catalyst istris-(dibenzylidene-acetone)-dipalladium(0).

The enolic hydroxy group of enolized compound IV is preferablyderivatized by a reaction with N-aryltriflimide to produce a triflate.

To improve the applicability of the new vitamin D analogs of theinvention for the above-described pharmacotherapeutic indications, thecompounds are usually processed to pharmaceutical compositions,comprising an effective amount of said vitamin D analog as the activeingredient in addition to a pharmaceutically acceptable carrier and/orat least one pharmaceutically acceptable auxiliary substance. Such acomposition may be delivered in a dosage unit form for oral, topical(dermal) or parenteral administration, comprising approx. 0.1 μg toapprox. 0.1 mg active ingredient per dosage unit. A composition fordiagnostic purposes may comprise, in addition to the vitamin D analog ofthe present invention, a compatible, non-toxic carrier and/or at leastone auxiliary substance.

A cosmetical composition may comprise, in addition to an effectiveamount (in the range of approx. 0.1 μg to. approx. 0.1 mg per dosageunit in a dosage unit form) of the vitamin D analog of the presentinvention, a cosmetically acceptable, non-toxic carrier and/or at leastone auxiliary substance.

Finally the invention relates to a method for the treatment andprophylaxis of a number of disease states including autoiummune diseases(including diabetes mellitus), acne, alopecia, skin aging (includingphoto-aging), imbalance in the immune system, inflammatory diseases suchas rheumatoid arthritis and asthma, as well as diseases related toabnormal cell differentiation and/or proliferation, in a warm-bloodedliving being, comprising administering to said being or treating saidbeing with a pharmaceutical composition as defined above in a quantityeffective for the intended purpose. Examples of such diseases arepsoriasis and other hyperproliferative skin diseases.

The present invention also relates to the use of the abovepharmaceutical compositions for the treatment of solid, skin and bloodcancers, in particular of blood cancers such as leukaemia, of breast andcolon cancer, and of skin cancers such as melanoma and squamous cellcarcinoma.

The above-defined cosmetical compositions, in particular selected fromthe group consisting of creams, lotions, ointments, liposomes and gels,can be used for the treatment and prevention of a number of skindisorders, such as inadequate skin firmness or texture, insufficientskin hydration, wrinkles and insufficient sebum secretion.

The invention will now be described in greater detail with reference tothe following specific Examples.

The following abbreviations are used in the examples:

THF=tetrahydrofuran

TBDMS=tert.-butyl dimethyl silyl

DMF=N,N-dimethylformamide

Pd₂dba₃=tris-(dibenzylideneacetone)dipalladium (0)

Examples

The TBDMS protected compound (IIIa) wherein X′=Br is dissolved in dryTHF, cooled to −78° C. and treated with 2 eq. of tert. C₄H₉Li. After 30minutes a solution of 1 eq. ZnCl₂ in dry THF is added and stirring iscontinued another 15 minutes. A solution of 1.1 eq. of a compound IVwherein R₄ is CF₃SO₃—, 5 mol % of the catalyst Pd₂dba₃ and 10 mol % ofAs₃P in dry DMF is introduced by means of double ended needle technique.The mixture is stirred at room temperature until the catalyst hascompletely dissolved. The progress of the reaction is monitored bythin-layer-chromatography. The reaction mixture is worked up byflash-chromatography. Complete deprotection of the hydroxy groups iscarried out by treatment of the obtained product with an ion exchangeresin, such as DOWEX-50 W, in methanol.

The following compounds of formula (I) have been prepared in thismanner:

Comp. Yield No. R₁ R₂ R₃ (%) 1 CH₂CH₂OH H (CH₂)₃—C(CH₃)₂OH 79 2 OH H(CH₂)₃—C(CH₃)₂OH 51 3 H CH₂OH (CH₂)₃—C(CH₃)₂OH 82 4 H CH₂CH₂OH(CH₂)₃—C(CH₃)₂OH 88 5 CH₂OH H (CH₂)₃—C(CH₃)₂OH 57

¹H-NMR data (CDCl₃,δ) of compounds no.s 1-5:

400 mHz ¹H-NMR (CDCl₃,δ): 0.76 (C₁₈H₃; 3H; s); 1.00 (C₂₁H₃; 3H, d); 1.22(C₂₆,₂₇H₃, 6H; S); 2.27 (C₁₁H₂, 2H, b); 2.58 (C₁₄H; 1 H, b); 2.83 (Φ-CH₂—C, 2x; 4H, t); 3.85 (2x Φ-C—CH₂—O, 4H, t); 5.64 (C₉H; 1H, b); 6.92 (A,1H, s); 6.93 (B; 2H, s).

400 mHz ¹H-NMR (CDCl₃, δ): 0.73 C₁₈H₃; 3H,s); 0.99 (C₂₁H₃, 3H, d); 1.19(C_(26,27)H₃; 6H,s); 2.23 (C₁₁H₂, 2H, b); 2.48 (C₁₄H, 1H, b); 5.58 (C₉H,1H, b); 6.16 (B, 2H, s); 6.20 (A, 1H, s); 8.53 (2xΦ-OH, 2H, s).

400 mHz ¹H-NMR (CDCl₃, δ): 0.77 (C₁₈H₃; 3H, s); 1.00 (C₂₁H₃, 3H, d);1.21 (C_(26,27)H₃; 6H, s); 2.58 (C₁₄H, 1H, b); 4.65 (Φ-CH₂—O, 2H, s);5.63 (C₉H; 1H, b); 7.18 (Φ-H, 2H, t); 7.26 (Φ-H, 2H, b).

400 mHz ¹H-NMR (CDCl₃, δ): 0.76 (C₁₈H₃, 3H, s); 1.00 (C₂₁H₃; 3H, d);1.22 (C_(26,27)H₃, 6H, s); 2.60 (C₁₄H, 1H, b); 2.84 (Φ-CH₂—C, 2H, t);3.85 (Φ-C—CH₂—O, 2H, t); 5.62 (C₉H, 1 H, b); 7.14 (Φ-H, 4H, b).

400 mHz ¹H-NMR (CDCl₃, δ): 0.76 (C₁₈H₃, 3H, s); 1.00 (C₂₁H₃, 3H, d);1.22 (C_(26,27)H₃, 6H, s); 2.59 (C₁₄H, 1H, b); 4.64 (2x Φ-CH₂—O, 4H, s);5.65 (C₉H; 1H, b); 7.11 (2x B; 2H, s); 7.18 (A, 1H; s).

Affinity Towards the Intracellular Vitamin D Receptor

The aromatic vitamin D analogs were dissolved in ethanol inconcentrations ranging from 10⁻¹⁴ to 10⁻⁶ M. The affinity towards thecalf thymus intracellular vitamin D receptor (VDR) was determined in abiological in vitro assay and compared to that of calcitriol. In thisassay, labelled ³H-calcitriol, which is specifically bound to the VDR,is replaced by the aromatic vitamin D analog. Compounds 5 and 3 haveeven higher affinities (resp. 6 and 3 times) than calcitriol itselftowards the highly selective VDR. Also analogs 4 and 1 demonstrate highVDR affinities. A high VDR affinity is indicative for biologicallyactive substances.

The compounds with the highest affinities were therefore further testedin vitro.

Affinity to Human Blood Vitamin D Binding Protein

Vitamin D binding protein (DBP) is the specific carrier for vitamin Dand its metabolites in blood. The biological activity of vitamin Danalogs depends on their binding to DBP. Strong binders will havereduced access to the VDR. Weak binders are rapidly metabolized, whichis a favourable aspect in topical application.

DBP is purified from total human serum. In the assay, DBP is incubatedwith ³H-calcitriol and calcitriol or one of the aromatic vitamin Danalogs. To this purpose, the vitamin D analogs are dissolved in ethanolin concentrations ranging from 10⁻¹¹ to 2.5×10⁻⁶.

The percentage bound/unbound ³H-calcitriol is then calculated. Compounds5, 4 and 3 (in order of binding) are relatively good binders, binding afactor 2-10 less than calcitriol itself. These binding capacities showthat the compounds might have systemic actions, i.e. they might beactive when administrated orally or given by injection.

What is claimed is:
 1. A vitamin D compound of formula (I):

wherein: R₁ is a hydrogen atom or a substituent selected from the groupconsisting of OH, CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH, OCH₃, OCH₂OH, OCH₂CH₂OHand OCH₂CH₂CH₂OH; R₂ is a hydrogen atom or a substituent selected fromthe group consisting of OCH₃, OCH₂OH, CH_(m)(CH₂OH)_(n)(CH₂CH₂OH)_(p),(CH₂)_(q)OH and O(CH₂)_(r)OH; wherein: m is 0 or 1; p, q, and n areindependently 0, 1, 2 or 3; r is 1, 2 or 3; and m+n+p=3; with theproviso that at least one of R₁ and R₂ contains at least one OH groupand; with the proviso that both R₁ substituents are the same; R₃ is astraight or branched, saturated or unsaturated aliphatic hydrocarbonhaving from 6 to 13 carbon atoms which may be substituted with one ormore substituents selected from the group consisting of hydroxy andfluoro.
 2. The compound of formula (I) as claimed in claim 1, wherein R₃is —(CH₂)₃—C(CH₃)₂OH.
 3. The compound of formula (I) as claimed in claim2, wherein: (a) R₁ is a hydrogen atom and R₂ represents CH₂CH₂OH,CH₂CH₂CH₂OH, CH(CH₂CH₂OH)₂, C(CH₂CH₂OH)₃ or C(CH₂CH₂OH)₂CH₂OH; or (b) R₁is OH, CH₂OH or (CH₂)₂OH and R₂ represents hydrogen, OH, OCH₃, orO(CH₂)_(r)OH, wherein r has the value 1, 2 or
 3. 4. The compound offormula (I) as claimed in claim 1, wherein R₁ is hydroxy, CH₂OH orCH₂CH₂OH, and R₂ represents hydrogen, CH₂OH or CH₂CH₂OH.
 5. A method forpreparing the vitamin D compound claimed in claim 1, which methodcomprises reacting, under the influence of an alkyl lithium compound, azinc halogenide, and a transmetallation catalyst, a compound of formula(IV):

wherein: R₃′ is a straight or branched, saturated or unsaturatedaliphatic hydrocarbon having from 6 to 13 carbon atoms which may besubstituted with one or more substituents selected from the groupconsisting of protected hydroxy and fluoro; and R₄ is a protectedhydroxy group; with a compound of formula (IIIa):

wherein: X′ is halogen; and R₁′ and R₂′ are the hydroxy protected formsof groups R₁ and R₂ defined in claim 1; and subsequently deprotectingthe hydroxy groups of R₁′ and R₂′.
 6. The method of claim 5, wherein X′is bromine.
 7. The method of claim 5, wherein the zinc halogenide iszinc chloride.
 8. A pharmaceutical composition comprising: apharmaceutically effective amount of at least one compound of formula(I) defined according to claim 1, and at least one pharmaceuticallyacceptable carrier, at least one pharmaceutically acceptable auxiliarysubstance, or a combination thereof.
 9. A method for preparing thepharmaceutical composition claimed in claim 8, wherein the methodcomprises combining said pharmaceutically effective amount of at leastone compound of formula (I) with said at least one pharmaceuticallyacceptable carrier, said at least one pharmaceutically acceptableauxiliary substance, or a combination thereof.